3. REAGENT PREPARATION - LAB Flashcards
Where can you find instructions for preparing reagents and quantities used?
Written laboratory protocol
What should be documented and matched to test performance in amplification methods?
Lot numbers and working stocks of probes and primers
Why should probe sequences used for a given test be recorded?
Because probes for linkage analysis and array technology are periodically updated
What is the purpose of an RNA primer in vivo?
Initiates DNA replication
What is the purpose of RNA primers in vitro (synthetically made primers)?
Used in DNA amplification
What is a critical component of PCR procedures?
Primers
What is the lyophilized form of a primer?
freeze dried or powdered form
What information is included in the primer product?
Quality, Method of purification, Molecular weight, Number of micrograms of dried primers
What is the typical length of oligonucleotide primers in base pairs?
18-25 or 18-30
What do probes bind to directly?
Target sequence
What is the purpose of probes?
Detection of target sequence or hybridization techniques
In what forms are probes supplied?
Solution or dried form
How should new working stocks of probes be treated?
As new reagent lots
What needs to be done to dried form probes or primers?
Dissolve in solvent to make stock solution
What should be done with stock solution to make a working solution?
Dilute it
What are analyte-specific reagents (ASRs)?
Probes, primers, antibodies
What does analyte-specific reagents detect?
Presence or absence of a particular substance
Where are analyte-specific reagents used?
Tissue typing, infectious disease, PCR
What are in vitro diagnostic (IVD) devices?
Instruments or systems for diagnosis
What are IVD reagent sets used for?
Collecting, preparing, or examining specimens
How are IVD devices classified?
CLASS I, CLASS II, CLASS III
Reagent categories
ASRs
IVD
IVAT
RUO IUO
This category is intended to accommodate the use of promising high complexity technologies facing long clearance processes
In vitro analytical test (IVAT) reagents
are not intended for diagnostic use
Research Use Only (RUO)
can be used in patient samples with proper institutional review and informed consent
Investigational Use Only (IUO)
reagents are used to gather data that may result in the advancement of a product’s status
Research Use Only (RUO) and Investigational Use Only (IUO) reagents
What is the first step in making reagents and buffers?
Go through the protocol for the experiment and write down all the solutions used.
What should you decide about the solution concentration?
Decide whether to make diluted or concentrated solutions.
What should you check regarding chemical availability?
Find out if the chemicals you need are available in the lab.
What should you check for safety information?
MSDS (Material Safety Data Sheet)
What label should you look for on hazardous materials?
Hazardous material classification label
Where should you read for warnings about the reagent?
Reagent bottle/container
What should you do if there are no warnings on the label?
Look up the material in the Merck Index
What if the label is in another language or you can’t find a listing?
Consult the manufacturer
What is a concern during the analytical phase of work?
Exposure to various hazardous reagents
What neurotoxin is used for protein and sequencing gels?
Acrylamide
What mutagen intercalates in DNA and is used to label nucleic acids?
Ethidium bromide (EtBr)
What highly corrosive substance can burn skin and should be used in a fume hood?
Phenol
What reagent, used to inhibit proteinases, can be fatal if swallowed or absorbed through the skin?
Phenylmethyl-sulfonate fluoride (PMSF)
What detergent is a burning powder and extremely irritating to nasal passages?
Sodium dodecyl sulfate (SDS)
What method is used for sterilizing most buffers?
Autoclave
What material are tips and microcentrifuge tubes made of for autoclaving?
High-density polyethylene
What should be avoided when autoclaving tips and microcentrifuge tubes?
Proteins
What should you check for in solutions before autoclaving?
Acidity or alkalinity
What substances should not be autoclaved?
Corrosives, solvents, volatiles, liquids with bleach/formalin/glutaraldehyde, buffers with detergents (e.g., 10% SDS), heat-labile ingredients, DTT, and β-mercaptoethanol
Storage of buffers in room temperature
Detergents
Ethanol
Buffers
Concentrated solutions
Acid and bases
Storage of buffers and solutions in 4C
Bacterial cultures
Buffers
DNA
Media
PCR products
Serum
Storage of buffer and solutions in -20c
Enzymes
RNA
Storage of buffer and solutions in -70c
Frozen bacterial cultures
Lipids
